A single nucleotide primer extension assay to detect the APC I1307K gene variant

Abstract

Adenomatous polyposis coli (APC) is a tumor suppressor gene important in colorectal tumorigenesis. A genetic variant of APC, I1307K, results from a T-to-A transversion at nucleotide 3920 which converts the wild-type sequence to a homopolymer tract (A(8)). The I1307K alteration is not itself oncogenic, but creates a hypermutable region (A(8)) that is prone to frame-shift mutations. The APC I1307K variant occurs in approximately 6% of the Ashkenazi Jewish population and is reported to approximately double an individual's risk for colorectal cancer. Here we describe a single nucleotide primer extension assay for the detection of the APC I1307K mutation. Following PCR amplification, nucleotide 3920 of the APC gene is directly sequenced using single nucleotide primer extension technology. The assay is in a multiplex format allowing simultaneous forward and reverse sequencing of the I1307K variant, which provides an internal, independent confirmation of each testing result. The assay was validated against 60 samples previously characterized by an allele-specific oligonucleotide (ASO) hybridization assay, with 100% concordance of results. Compared to the ASO assay, this single nucleotide primer extension assay requires significantly less technical time to perform, and has a greatly increased throughput capacity. The single nucleotide extension assay provides a highly sensitive and specific assay to identify individuals with the APC I1307K gene variant who may benefit from increased colorectal screening.

Figure 1.

Schematic of the single nucleotide primer extension assay. The boxes represent the PCR product, the sequence surrounding the I1307K variant is demonstrated. The forward (21-base oligomer) and reverse (24-base oligomer) SNaPshot primers are indicated by arrows. A wild-type and mutant allele are depicted demonstrating the fluorescent ddNTP which extends each primer.

Figure 2.

Examples of SNaPshot results. Capillary electrophoresis pherograms, x axis is size in bases, y axis is fluorescence intensity. The orange peaks represent the internal size standard. The arrows labeled “F” indicate SNaPshot products resulting from extension of the forward primer, sizing at 26 bases. The arrows labeled “R” indicate SNaPshot products resulting from extension of the reverse primer, sizing at either 30 or 31 bases. The addition of a thymidine (T) ddNTP to a primer results in a red-colored product/peak, the addition of an adenine (A) ddNTP results in a green-colored peak. A: Wild-type result demonstrating a red (T) peak at 26 bases resulting from extension of the forward primer and a green (A) peak at 30 bases resulting from extension of the reverse primer. B: Heterozygous mutant result demonstrating superimposed green (A) and red (T) peaks at 26 bases resulting from extension of the forward primer. The green (A) peak at 30 bases and the red (T) peak at 31 bases result from extension of the reverse primer. The green and red reverse primer peaks travel at different sizes due to the effect of the fluorophore on migration. C: Homozygous mutant result demonstrating a green (A) peak at 26 bases resulting from extension of the forward primer and a red peak at 31 bases resulting from extension of the reverse primer.

Figure 3.

Results from the ASO assay. PCR products were slot-blotted onto two nylon membranes and probed with either a wild-type or mutant radioactive probe. After incubation and washing, the membranes were exposed to film and the results were compared. Lane 1 is a no template control. Lanes 2, 4,and 5 hybridized with both the wild-type and mutant probes and are thus interpreted as heterozygous positive for the APC I1307K variant. Lanes 3 and 6 hybridized with the wild-type probe only and are thus interpreted as wild-type (negative for the I1307K variant).